Assessment of bacterial viability status by flow cytometry and single cell sorting

Caron, G. Nebe-von; Stephens,; Badley,

doi:10.1046/j.1365-2672.1998.00436.x

Cited by 134 publications

(40 citation statements)

References 23 publications

(28 reference statements)

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“…Viability is a gradient from actively growing cells to completely dead cells with disruption of vital functions (5). The different viable/dead methods applied use different criteria for viable/dead measurements (Table 1) (5,6,11,24,29).…”

Section: Use Of Ema-pcr To Study Survival Under Decontamination and Amentioning

confidence: 99%

“…Viable/dead determinations are key issues in many aspects of biological research. The current technologies addressing this important issue have severely limited application ranges (4,5,14,18,19). There are for instance no approaches enabling accurate viable/dead quantifications in mixed cell populations (2, 13).…”

mentioning

confidence: 99%

“…Viable/dead analyses of pure monocultures are not new or novel. A wide range of different approaches exist (2,5,10,12,25,26). Methods for direct quantitative analyses of complex samples, however, are still lacking.…”

mentioning

confidence: 99%

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Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

Rudi¹,

Moen²,

Drømtorp³

et al. 2005

Appl Environ Microbiol

345

286

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The distinction between viable and dead cells is a major issue in many aspects of biological research. We have developed a novel concept for quantification of viable and dead cells in complex samples. The viable/dead stain ethidium monoazide (EMA) is used in combination with realtime PCR to inhibit amplification of DNA from dead cells that have taken up EMA (Fig. 1). Viable/dead determinations are key issues in many aspects of biological research. The current technologies addressing this important issue have severely limited application ranges (4,5,14,18,19). There are for instance no approaches enabling accurate viable/dead quantifications in mixed cell populations (2, 13).Real-time PCR is the most widely applied technology for direct quantification of cells in mixed samples. Real-time PCR is increasingly being used for direct detection and quantification of pathogens in foods and environmental or clinical samples. Still, a major obstacle with PCR diagnostics is how to distinguish between DNA from viable and dead cells. Intact DNA can be present although the organisms are dead. This is particularly relevant for pathogens subjected to killing treatments such as disinfections or antibiotics. Even greater challenges are encountered with organisms such as Campylobacter jejuni that have specific growth requirements and may enter a state where it is viable and infectious but not cultivable. The lack of viable/dead differentiation has been a serious limitation for the implementation of DNA diagnostics in routine applications (15,19,22).We have recently used ethidium monoazide (EMA)-PCR for qualitative DNA-based viable/dead differentiation of bacteria in pure monoculture models (21). Viable/dead analyses of pure monocultures are not new or novel. A wide range of different approaches exist (2,5,10,12,25,26). Methods for direct quantitative analyses of complex samples, however, are still lacking. Solving these analytical problems would be a major technological breakthrough. We discovered during the work with the monoculture models that EMA-PCR has this potential. Thus, the aim of the present work was to use EMA-PCR to show that it is possible to develop quantitative assays for specific viable and dead bacteria in complex samples with mixed bacterial populations. We developed the assay for the major food-borne pathogenic bacterium C. jejuni due to the apparent need for new viable/dead diagnostics of this bacterium. The conditions analyzed include detection in mixed and natural samples, survival in foods, and after disinfection and antibiotic treatments. This knowledge is crucial both for diagnostics and in the control of C. jejuni.A dynamic range of more than 4 log 10 was obtained for the EMA-PCR viable/dead assay. We were able to reliably quantify the fraction of viable C. jejuni under all conditions tested, including complex samples with mixed populations. This is to our knowledge the first time that quantitative viable/dead information has been obtained from specific bacteria in mixed populations. C. jejuni was used as...

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Section: Use Of Ema-pcr To Study Survival Under Decontamination and Amentioning

confidence: 99%

mentioning

confidence: 99%

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Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

Rudi¹,

Moen²,

Drømtorp³

et al. 2005

Appl Environ Microbiol

345

286

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“…Once we can assure biosafety by adding formaldehyde before acquiring results in the flow cytometer, further containment would not be required. Microbiological use of a flow cytometer should be considered (even if only for screening of drug resistance to help avoiding the spread of drug-resistant tuberculosis) (1,4,5,16). We could confirm the feasibility of use of flow cytometry with clinical samples of M. tuberculosis in 24 h.…”

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confidence: 49%

Comparison of Flow Cytometric and Alamar Blue Tests with the Proportional Method for Testing Susceptibility of Mycobacterium tuberculosis to Rifampin and Isoniazid

et al. 2004

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The performance of flow cytometry and the microplate Alamar Blue assay in determining susceptibility of Mycobacterium tuberculosis was assessed by testing 150 Brazilian isolates. The overall agreement was 97.3 and 98% for isoniazid and 94.7 and 100% for rifampin by flow cytometry and MABA, respectively. This study was entirely done in a developing country.Many developing countries have serious difficulties obtaining drug susceptibility information for Mycobacterium tuberculosis isolates for financial or technical reasons. Treatment of tuberculosis without susceptibility information increases the risk of treatment failure and of the spread of resistant strains as well as the risk of the development of resistance to additional drugs (2,8).The commonly used agar proportion method for mycobacterial susceptibility testing requires a 3-to 4-week period of incubation before a pattern of susceptibility is established (3, 9). Flow cytometry that relies on fluoresceine diacetate (FDA) for detection has been used to perform susceptibility testing of M. tuberculosis and can yield results within 24 h. The inhibition by rifampin (RIF) and isoniazid (INH) of the ability of viable M. tuberculosis to hydrolyze FDA can be measured by flow cytometry. In addition, multiplication of the mycobacteria is not required (10,11,15).The microplate Alamar Blue assay (MABA) (7) has been reported to show very good correlations with the proportional and BACTEC methods. MABA is a resarzurin-based oxidation-reduction indicator which measures colorimetric drug MICs for M. tuberculosis for up to 7 days. In this study, we compared the flow cytometric and MABA tests with the standard proportional method to assess INH and RIF susceptibilities of 150 clinical isolates from a community in Rio de Janeiro, Brazil; 100 isolates were susceptible to both drugs, 50 were resistant to INH, and 37 of these 50 were also RIF resistant (multidrug resistant).The proportional method was performed according to the method of Canetti, Rist, and Grosset (3). The results obtained by the proportional method were used as a reference to compare the results of flow cytometry and MABA.For mycobacterium preparation, each isolate was grown in two Löwenstein-Jensen tubes (Difco, Detroit, Mich.) at 37°C in aerobic conditions for 30 days. After incubation, colonies were suspended in Middlebrook 7H9 medium (Difco) directly from solid medium and adjusted to a no. 1 McFarland standard (ϳ3 ϫ 10 7 CFU/ml).Flow cytometric susceptibility testing was performed according to the method of Norden et al. (11). The only modification was implemented to assure bacterial inactivation before analysis with an XL-MCL flow cytometer (Coulter, Miami, Fla.). In a previous study (data not published), we showed that M. tuberculosis cells can be killed by formaldehyde at a final concentration of 10% for 1 h.Final drug concentrations were 0.5, 1.0, and 2.0 g/ml for RIF and 0.1, 0.2, and 0.3 g/ml for INH. For each isolate the relative fluorescence value of each drug-containing sample was divided by the rela...

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“…Flow cytometry is a very sensitive, efficient assay and does not require any cell culturing or enrichment procedures. For a flow cytometric assay, fluorescent dyes are used as sensitive indicators to detect and distinguish the cell viability in many cell types (3,5,10). Propidium iodide (PI), the fluorescent stain used in this study, is a nucleotide binding dye and theoretically only enters cells with damaged membranes; hence it is generally used to label dead cells (2,13).…”

Section: Introductionmentioning

confidence: 99%

Determine the Potency of BCG Vaccines by Flow Cytometer

Yang

Tsai

Cheng

2011

Biotechnology & Biotechnological Equipment

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Assessment of bacterial viability status by flow cytometry and single cell sorting

Cited by 134 publications

References 23 publications

Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

Comparison of Flow Cytometric and Alamar Blue Tests with the Proportional Method for Testing Susceptibility of Mycobacterium tuberculosis to Rifampin and Isoniazid

Determine the Potency of BCG Vaccines by Flow Cytometer

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